A Crosslinked Collagen Hydrogel Matrix Resisting Contraction to

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A Crosslinked Collagen Hydrogel Matrix Resisting Contraction to Facilitate Full-thickness Skin Equivalents Christian Lotz, Freia F. Schmid, Eva Oechsle, Michael Monaghan, Heike Walles, and Florian Kai Groeber-Becker ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017

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ACS Applied Materials & Interfaces

A Crosslinked Collagen Hydrogel Matrix Resisting Contraction to Facilitate Full-thickness Skin Equivalents ‡Christian Lotz1, ‡Freia F. Schmid2, Eva Oechsle2, Michael G. Monaghan3,4,5, Heike Walles1,2, Florian Groeber-Becker2* ‡Both authors contributed equally 1 Department Tissue Engineering & Regenerative Medicine (TERM), University Hospital Würzburg; Würzburg 97070, Germany

2 Translational Center Würzburg ´Regenerative therapies in oncology and musculoskeletal diseases` Würzburg branch of the Fraunhofer Institute for Interfacial Engineering and Biotechnology; Würzburg 97070, Germany

3. Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart 70569, Germany

4.Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland

5. Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland

KEYWORDS Regenerative medicine, Tissue Engineering, Collagen, Crosslinking, Alternatives to animal testing, Skin Grafts 1 ACS Paragon Plus Environment


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ABSTRACT

Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as non-animal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically crosslink the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a non-cytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), crosslinking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg PEG-SG per mg collagen led to a viability comparable to noncrosslinked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Crosslinked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-crosslinked collagen hydrogels. Remarkably, during 21 days, crosslinked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50 %. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the

equivalents

and

culturing

these

equivalents

at

an

air-liquid-interface.

Immunohistochemical stainings of the crosslinked model revealed well defined epidermal layers including an intact stratum corneum and a dermal part with homogenously distributed human dermal fibroblasts. These results indicate that crosslinking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.

2 ACS Paragon Plus Environment

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INTRODUCTION The skin is the primary interface between the human body and the environment it encounters. Hence, it can be prone to extensive tissue damage and is exposed to numerous chemicals that can lead to adverse health effects. If the integrity of skin becomes impaired, plain gauze is the most commonly used wound dressing in hospitals for small injuries that only effect the epidermis or just superficial parts of the dermis. For larger or deeper wounds, the biological mechanism for wound closure changes from regeneration to contraction of the wound, resulting in the formation of scar tissue and loss of function. Full-thickness injuries with a surface area larger than 1 cm² require a skin graft to prevent extensive scarring 1. The current ‘gold standard’ for skin replacement is an autologous full- or split-thickness skin graft 2-3

. However, this method is limited by the availability of healthy tissue and inflicts secondary

wounds and additional pain for the patient. Therefore, tissue engineered skin equivalents are being developed as skin substitute in regenerative medicine 4-6. Aside from clinical applications tissue engineered skin models can be used as pre-clinical test models for risk and efficacy assessment as an alternative to traditional animal models. Species-specific variances in skin architecture and metabolism have led to an increasing demand for alternatives to animal models. Additionally, international and European regulations have become more stringent regarding product safety and the use of animal-free alternative test methods 7. Therefore, numerous efforts have been attempted to provide alternatives to animal models for dermal toxicity testing in recent years 8. Currently, two different versions of artificial skin models are available. Reconstructed human epidermis consists exclusively of a multi-layered epidermal construct, whereas full-thickness skin models constitute an epidermis grown upon a dermal layer 9. Although reconstructed human epidermis is a promising tool for toxicity testing, the lack of a dermal part limits the applicability of the models. Previous studies have shown that the crosstalk between the epithelium and the connective tissue regulates skin morphology and homoeostasis

10

.



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Additionally, there is experimental evidence that metabolic activity is reduced in the absence of a dermis

11-12

. With regard to the assessment of toxicological endpoints that require an

interaction with cutaneous enzymes e.g. genotoxicity, this lack of influential crosstalk can be problematic since it decreases the accuracy of test methods based on reconstructed human epidermis considerably

13

. Furthermore, skin grafts benefit from a dermal layer for wound

healing. The dermal layer increases the chance of graft integration up to 75% and provides growth factors and cytokines regulating wound healing 14. Hence, to improve on recapitulating human skin for in vivo and in vitro application, the use of tissue engineered skin including both skin layers is highly desirable. A key element of fullthickness equivalents is the matrix that is used to generate the dermal layer. Since the most abundant proteins within the dermis are collagen type I and type III, most skin models are generated using collagen hydrogels 15-17. However, a major pitfall of collagen-based hydrogels is their susceptibility to fibroblast-mediated contraction during culture that results in an epidermal layer that lacks attachment to the insert wall 18. In a similar way, skin substitutes can contract and degrade, reducing their efficacy and engraftment chances. Thus, hydrogel contraction impedes the standardization of full-thickness skin models and their reliable application for toxicity testing and the clinical application as skin implants. Several attempts have been made to overcome the contractibility of collagen hydrogels containing human dermal fibroblasts. Besides physical modification of collagen hydrogels by plastic compression 19, the chemical crosslinking of collagen has yielded in reduced collagen contraction

20-21

. Due to the cytotoxic properties of most crosslinking agents such as

glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, the crosslinking process is usually performed prior to cell seeding, after which remnants of the crosslinking reaction are washed out to facilitate cell culture

22-23

. However, these approaches limit the creation of

homogenous scaffolds that can be used in in vitro models and clinical applications. In order to create a crosslinked collagen hydrogel that can serve as a matrix for supporting dermal layer 4 ACS Paragon Plus Environment

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of full-thickness skin models, a crosslinker that can be directly incorporated into the matrix during cell seeding prior to hydrogel gelation is needed. Previously, such crosslinking approaches have included the use of microbial transglutaminase and multi-arm polyethylene glycol polymers of varying molecular weights, branching and functional terminations 24-26. In this study we explore the use of a four-arm polyethylene glycol with succinimydil glutarate (PEG-SG) terminated branches. Previously, PEG-SG was investigated as a crosslinking agent to crosslink collagen hydrogels during fibroblast incorporation delivering exogenous microRNA 26. This study hypothesizes that chemically crosslinking a collagen hydrogel harbouring fibroblasts can be achieved using PEG-SG without the induction of cytotoxic effects and that such a chemically modified matrix elicits a reduction of fibroblast-mediated contraction. Crosslinked collagen hydrogels were generated and evaluated in regard to their chemical and mechanical stability, ultrastructure, viability and contraction. Finally, crosslinked fullthickness skin equivalents were generated to demonstrate the capacity of the crosslinked collagen to facilitate skin tissue formation. MATERIAL AND METHODS Cell isolation Human dermal fibroblasts and human epidermal keratinocytes were isolated from foreskin biopsies of 2–5 year old donors with approval of the local ethics committee (approval number IGBZSF-2012-078) and after the confirmed consent of their guardians using a wellestablished protocol described previously

27

. Briefly, biopsies were washed, minced and

digested with dispase (Life technologies, Germany) to dissociate the epidermis from the dermis. Thereafter, the epidermis was trypsinized (Life technologies, Germany) and the dermis was digested with collagenase (Serva, Germany) to generate single cell suspensions. Crosslinking of collagen using four-armed polyethylene glycol succinimidyl glutarate and generation of dermal equivalents 5 ACS Paragon Plus Environment


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To generate crosslinked collagen hydrogels, type I rat tail collagen at 6 mg/ ml in 0.1 % acetic acid was mixed with gel neutralization solution (75 mM HEPES, 48 mM glucose, 268 mM NaCl; Sigma Aldrich, Germany) at a 1:1 volumetric ratio. For crosslinking, different concentrations of PEG-SG (molecular weight 10,000; JenKem technology, USA) were additionally added to the gel neutralization solution. Therefore the desired amount of PEG-SG was solubilized in ice cold gel neutralization solution and subsequently mixed with the collagen solution for 30 seconds at room temperature. The amount of PEG-SG was based upon the molar ratio of free collagen primary amine groups and the n-hydroxysuccinimide ester of PEG-SG. With the four-arm structure of PEG-SG, theoretically, one expects one mole of PEG-SG to react with four moles of free amines available in collagen type I. Therefore, 1 mg collagen was mixed with 0.5 mg or 1 mg PEG-SG resulting in a theoretical crosslinking of 50 % or 100 % of all free amine groups. A detailed calculation of the molar ratio of collagen primary amine groups and the n-hydroxysuccinimide ester of PEG-SG can be found in the supporting information. Following, a PEG-SG concentration theoretically sufficient to crosslink 50 % of all free amines will be termed PEG-SG50 and a PEG-SG concentration that can theoretically crosslink all free amines will be termed PEG-SG100. After mixing the gel neutralisation solution with PEG-SG and collagen type I solution the hydrogels were incubated for 10-15 minutes at 37 °C for completion of gelation. For the generation of dermal equivalents human dermal fibroblasts were added to the gel neutralization solution in the second passage of culture resulting in a final concentration of 1 x 105 cells/ ml in the dermal equivalent. A schematic of the crosslinking reaction is depicted in Figure 1A. Noncrosslinked collagen hydrogels were generated in a similar manner, but without the addition of the crosslinker. Transparency of collagen hydrogels The optical density of 200 µl acellular crosslinked, non-crosslinked hydrogels and nonassembled collagen solutions was determined by an absorption scan from 500-800 nm in a 966 ACS Paragon Plus Environment

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well plate using a spectrophotometer (Infinite 200M; Tecan, Switzerland). Transmission was calculated as a ratio of the incident light and absorbed light. Scanning Electron Microscopy (SEM) Hydrogels were fixed with 2.5 % glutaraldehyde in 50 mM cacodylate buffer for 48 hours at 4 °C. Samples were dehydrated in a series of increasing acetone concentrations, dried by critical point drying, sputtered with gold-palladium and examined with a field emission SEM JEOL JSM-7500F (JEOL, Germany). Transmission Electron Microscopy (TEM) Hydrogels were fixed at 4 °C for 48 hours in 2.5 % glutaraldehyde in 50 mM sodium cacodylate buffer and post-fixed using 2 % osmium tetroxide for 2 hours at 4 °C. Ultrathin sections were stained with 0.5 % aqueous uranyl acetate, dehydrated with ethanol and embedded in Epon 812. Sections were inspected with a TEM JEOL JEM-2100 microscope (JEOL, Germany). Mechanical characterization of hydrogels The mechanical properties of the hydrogels were assessed by performing a compression test with a materials testing machine (Zwick/Roell Z005; Zwick GmbH & Co KG, Germany) comprising a 2.5 kN load cell. Data were recorded by using the testing software testXpert II (Zwick GmbH & Co KG, Germany). After preparation the hydrogels were incubated for 24 hours at 37 °C. A cylindrical hydrogel sample with a diameter of 1.5 mm and a height of 10 mm was placed between the plates of the instrument. For the compression test a constant traverse speed of 1 mm/ min was conducted until a gap of 2 mm between traverse and crosshead was reached. A stress-strain curve was plotted during the measurement. The elastic compressive E-modulus of each sample was evaluated as a ratio of the stress and strain in the linear area of the stress-strain curve. Collagenase assay



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Resistance of the hydrogels to enzymatic digestion was evaluated using a collagenase assay. Hydrogels with a volume of 500 µl were incubated for 1 hour in 1 ml 0.1 M Tris–HCl (pH 7.4), containing 50 mM CaCl2 (Sigma Aldrich, Germany) at 37 °C. Subsequently, the TrisHCl solution was substituted for 1 ml bacterial collagenase solution (10 units/ mg collagenase type IV in 0.1 M Tris–HCl; Life Technologies, Germany). After incubation for 14 hours at 37 °C,

the

enzymatic

reaction

was

halted

by

the

addition

of

1 ml

0.25 M

ethylenediaminetetraacetic acid. Following vacuum dehydration, the remaining mass of the hydrogels was determined and normalized to the remaining mass of non-digested hydrogels. Viability assessment of dermal equivalents The viability of crosslinked and non-crosslinked hydrogels seeded with 1 x 105 cells/ ml human dermal fibroblasts after 21 days of culture was quantified by using a 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assay. Cell-seeded hydrogels were placed into 3 ml of MTT solution (1 g/ ml MTT; Sigma Aldrich, Germany). After incubation for 24 hours at 37 °C, the MTT solution was discarded and the resulting precipitate solubilized in 3 ml of 2-propanol for 24 hours at 4 °C. Subsequently, the solubilized formazan salt was quantified by measuring the absorbance at 570 nm using a spectrophotometer (Infinite 200M; Tecan, Switzerland). Additionally, the presence of lactate dehydrogenase (LDH), indicative of cell lysis, was measured by quantifying the enzyme activity of LDH using a commercially available cytotoxicity detection kit (Cytotoxicity Detection Kit PLUS, Roche, Germany). Crosslinked and non-crosslinked hydrogels seeded with 1 x 105 cells/ ml fibroblasts were prepared and cultured for 24 hours. As a positive control, hydrogels treated with a 1 % Triton X-100 solution for 3 hours at 37 °C were included in the analysis. For quantification of LDH, 100 µL of freshly prepared reaction mixture was added to 100 µL of the supernatant of each sample in an optically clear 96-well flat bottom microplate. After 30 minutes, reduction of a tetrazolium


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salt to a reddish formazan product indicative for LDH activity was quantified by measuring the absorbance at 492 nm using a spectrophotometer (Infinite 200M; Tecan, Switzerland). Contraction of dermal equivalents To examine the capability of human dermal fibroblasts to contract hydrogels a contraction study was performed for 21 days. After generation of crosslinked and non-crosslinked hydrogels with 2 x 105 cells/ ml the hydrogels were kept at 37 °C in 95 % humidity and 5 % CO2 atmosphere. Culture medium was replaced twice a week. Surface area was analysed macroscopically after 1, 6, 9, 12, 14 and 21 days of culture. Generation of full-thickness skin models Skin models were generated on a polycarbonate membrane of respective cell culture inserts (diameter 0.59 cm², pore size 8 µm; Brand, Germany). Human dermal fibroblasts were resuspended in gel neutralisation solution according to the generation of the dermal part with or without PEG-SG. A volume of 150 µl collagen and 150 µl gel neutralization solution per insert was used, resulting in a fibroblast density of 1 x 105 cells/ml. After incubation at 37 °C for 24 hours in Dulbecco’s Modified Eagle Medium (Invitrogen, Germany) supplemented with 10 % fetal calf serum (Lonza Group Ltd., Switzerland) human epidermal keratinocytes were seeded onto dermal equivalents. Keratinocytes were applied with a cell density of 5 x 105 cells/cm², in 150 µL EpiLife® supplemented with 0.2 % v/v bovine pituitary extract, 1 µg/ml recombinant human insulin-like growth factor-I, 0.18 µg/ml hydrocortisone, 5 µg/ml bovine transferrin, 0.2 ng/ ml human epidermal growth factor (all from Life Technologies, Germany) and 1.5 mM CaCl2 (Sigma-Aldrich, Germany). To ensure sufficient nutrient supply to the skin models, inserts were cultured in 6.5 ml medium per row in custom 6 well-plates (Brand, Germany). Medium was changed after 24 hours to EpiLife® air-liquid-interface medium

28

, additionally containing 73 µg/ml L-ascorbic acid 2-phosphate and 10 ng/ml

keratinocyte growth factor (both Sigma-Aldrich). Medium was replaced by fresh air-liquidinterface medium three times a week and models were cultured at 37 °C and 5 % CO2 in a 9 ACS Paragon Plus Environment


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humidified incubator. Full-thickness skin models were used for testing after 19 days of airlift culture. Histological analysis Following the respective culture periods, cell seeded hydrogels were fixed in Bouin’s fixation agent (Sigma-Aldrich, Germany) for 1 hour, washed in tap water for 2 hours and embedded in paraffin. Subsequently, histological cross-sections of 3 µm were generated. For an overview of general histological features, tissue slides were stained with hematoxylin and eosin (H&E). Immunohistochemical staining was used to visualise specific epitopes in native skin and skin equivalents. Immunohistochemical staining was performed with the Dako EnVisionTM Kit (Dako, Denmark) according to the manufacturer’s protocol. After blocking endogenous peroxidase with 3 % H2O2 for 5 minutes, the primary antibody cytokeratin 14 (dilution of 1:500; Sigma-Aldrich, Germany), cytokeratin 10 (dilution of 1:200; Dako, Denmark), filaggrin (dilution of 1:100; Biomol GmbH, Germany), involucrin (dilution of 1:150; Acris Antibodies GmbH, Germany), vimentin (dilution of 1:1000; Abcam, United Kingdom), Ki67 (dilution of 1:150; Dako, Denmark), was applied and incubated at 4 °C overnight. One drop of horse radish peroxidase-coupled secondary antibody was added per section and the slides were incubated for 30 minutes at room temperature. The sections were covered with substrate solution for 4-7 minutes at room temperature until a macroscopic staining was observable. To visualize the cell structure, counterstaining with hematoxylin was performed. After incubation in 2-propanol, the sections were covered with Isomount 2000 (Labonord, France). Determination of proliferation index To determine proliferative human epidermal keratinocytes in skin equivalents, crosssections were immunohistochemically stained with an antibody against Ki67. The proliferation index of keratinocytes in the stratum basale was determined by the number of


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Ki67 positive nuclei in a total number of 100 basal cells, which was set to 100 %. Three different sections of each sample were evaluated. Statistical analysis Statistical analysis was performed using one way ANOVA with Dunnett’s post-test using the appropriate control in each experiment as a reference. Values of p

A Crosslinked Collagen Hydrogel Matrix Resisting Contraction to

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